Trichoderma afroharzianum TRI07 metabolites inhibit Alternaria alternata growth and induce tomato defense-related enzymes

Identifying a viable substitute for the limited array of current antifungal agents stands as a crucial objective in modern agriculture. Consequently, extensive worldwide research has been undertaken to unveil eco-friendly and effective agents capable of controlling pathogens resistant to the presently employed fungicides. This study explores the efficacy of Trichoderma isolates in combating tomato leaf spot disease, primarily caused by Alternaria alternata. The identified pathogen, A. alternata Alt3, was isolated and confirmed through the ITS region (OQ888806). Six Trichoderma isolates were assessed for their ability to inhibit Alt3 hyphal growth using dual culture, ethyl acetate extract, and volatile organic compounds (VOCs) techniques. The most promising biocontrol isolate was identified as T. afroharzianum isolate TRI07 based on three markers: ITS region (OQ820171), translation elongation factor alpha 1 gene (OR125580), and RNA polymerase II subunit gene (OR125581). The ethyl acetate extract of TRI07 isolate was subjected to GC–MS analysis, revealing spathulenol, triacetin, and aspartame as the main compounds, with percentages of 28.90, 14.03, and 12.97%, respectively. Analysis of TRI07-VOCs by solid-phase microextraction technique indicated that the most abundant compounds included ethanol, hydroperoxide, 1-methylhexyl, and 1-octen-3-one. When TRI07 interacted with Alt3, 34 compounds were identified, with major components including 1-octen-3-one, ethanol, and hexanedioic acid, bis(2-ethylhexyl) ester. In greenhouse experiment, the treatment of TRI07 48 h before inoculation with A. alternata (A3 treatment) resulted in a reduction in disease severity (16.66%) and incidence (44.44%). Furthermore, A3 treatment led to improved tomato growth performance parameters and increased chlorophyll content. After 21 days post-inoculation, A3 treatment was associated with increased production of antioxidant enzymes (CAT, POD, SOD, and PPO), while infected tomato plants exhibited elevated levels of oxidative stress markers MDA and H2O2. HPLC analysis of tomato leaf extracts from A3 treatment revealed higher levels of phenolic acids such as gallic, chlorogenic, caffeic, syringic, and coumaric acids, as well as flavonoid compounds including catechin, rutin, and vanillin. The novelty lies in bridging the gap between strain-specific attributes and practical application, enhancing the understanding of TRI07’s potential for integrated pest management. This study concludes that TRI07 isolate presents potential natural compounds with biological activity, effectively controlling tomato leaf spot disease and promoting tomato plant growth. The findings have practical implications for agriculture, suggesting a sustainable biocontrol strategy that can enhance crop resilience and contribute to integrated pest management practices.


Isolation of Trichoderma from tomato rhizosphere
Soil samples from the rhizosphere of tomato plants in healthy growth zones were obtained.The cultivation of Trichoderma and the utilization of selective medium, namely TME 49 and TSM 50 , were employed in this study.The fungal cultures obtained by the single spore isolation method 51 were purified and thereafter maintained on PDA slants for additional experimental procedures.

Morphological and molecular identification of isolated Alternaria and Trichoderma
The identification of isolated fungi was conducted based on their morphological and molecular features.The molecular typing of the pathogen was conducted using the internal transcribed spacer (ITS) region.The primer sequences employed for the ITS region were ITS1 and ITS4, and the PCR reactions were conducted using the methodology described in a previous study 52 .To identify the bioagent, Carbone et al. 53 and Abdelkhalek et al. 54 , www.nature.com/scientificreports/employed three molecular markers for Trichoderma isolate: the ITS region, the translation elongation factor alpha 1 (tef1) gene, and the RNA polymerase II subunit (rpb2) gene.In the PCR reactions, 1 µL of each primer pair (10  pmol), 20 µL of TOPsimple™ PCR PreMIX-nTaq (Enzynomics Inc., Yuseong-gu, Daejeon, Republic of Korea), 3 µL of fungus DNA, and 26 µL of molecular-grade water were combined.Cycling conditions were implemented using a thermal cycler TC-PRO (Boeco, Germany): an initial step at 94 °C for 4 min, followed by 35 cycles at 95 °C for 1 min (55 °C for ITS; 45 s, 62 °C for rpb2; 45 s, 57 °C for tef1; 60 s), and 72 °C each for 1 min, with a final extension step at 72 °C55 .The PCR amplification samples underwent sequencing at Macrogen Inc. (Seoul, Korea), and the NCBI-BLAST tool was employed to conduct a comparative analysis between the obtained sequences and those present in the GenBank database, therefore verifying their identification.The alignment of nucleotide sequences was performed utilizing the MEGA 11 program 56 , which was employed for editing and curating the sequences to generate phylogenetic trees.The potential phylogenetic relationships among A. alternata isolates and Trichoderma species were determined using the maximum-likelihood (MLL) technique for ITS, tef1 and rpb2 genetic markers sequence data, employing MEGA 11.Gap/missing data treatment involved adjusting the entire deletion, and the initial MLL tree was automatically modified.The MLL trees obtained underwent evaluation through bootstrap analysis with 1000 replications 56,57 .

Effect of Trichoderma isolates against the radial growth of Alternaria pathogen in vitro
A total of six isolates belonging to Trichoderma spp.were examined in this section.These isolates were obtained from the rhizosphere soil of tomato plants.The objective was to evaluate their efficacy against the pathogen responsible for tomato leaf spot.Various methods were employed to assess the efficiency of these isolates.

Dual culture technique
The efficacy of six Trichoderma isolates in controlling the pathogen associated with tomato leaf spot was evaluated using a dual-culture methodology 58,59 .The isolated pathogen and Trichoderma culture mycelial with a diameter of 5 mm were positioned in the Petri plates across from one another at an equal distance from the edge.On one side of the PDA-containing petri dish, antagonists were placed.A disc from the pathogen culture was positioned on the other side of the Petri plates and incubated at 25 °C ± 2 60 .Petri dishes inoculated with fungal pathogen were only used as control.The experiment trail was repeated 3 times.The inhibition percentage of pathogen growth was calculated according to following formula: Inhibition (%) = [(C − T)/C] × 100 , where C represents the control and T represents the development of the pathogen in the treated plates with antagonistic isolates 61 .

Ethyl acetate extract of Trichoderma isolates and its antifungal activity
Trichoderma isolates were grown on 100 mL of potato dextrose broth (PDB).The inoculated cultures were incubated at 25 °C ± 2 for 2 weeks under stirring (100 rpm) in INCU-SHAKER shaking incubator (Benchmark Scientific, Inc., Sayreville NJ, USA).
After 2 weeks, the fungal cultures were centrifuged at 6000 rpm for 10 min, then to ensure the filtration process, spores and mycelia of Trichoderma isolates were removed from PDB culture by filtration with Whatman no.1 filter paper.The rested broth media (culture filtrate) were used to extract the antagonistic metabolites by mixing the culture filtrate with ethyl acetate (1:1 v/v).Two clear immiscible layers were developed after the mixture had been thoroughly stirred for 10 min and allowed to sit for 5 min 62 .Using a separating funnel, the top layer of the solvent, which included the extracted biomolecules, was separated.The ethyl acetate extract was concentrated to small volume by evaporating the solvent in a rotary evaporator under vacuum 63 .After the ethyl acetate evaporated, brown gum was presented.After that, the crude extract was kept at 4 °C.
The antifungal activity of the prepared extract against the pathogen was evaluated using the poisoned food technique.After the extract was reconstituted (4 mg/mL) in dimethyl sulfoxide (DMSO) in 20 mL PDA, a 5 mm disc of the pathogen was put in the center of each Petri plate.Once the control plate reached the margins, the radial growth of pathogen was measured.The inhibitory effect were calculated compared to the control 64 .

Volatile compounds emitted from Trichoderma
The sealed plate technique 65 was used to examine the effectiveness of volatiles generated from Trichoderma isolates in inhibiting the growth of leaf spot pathogen.A mycelial plug (0.5 cm in diameter) was plugged from a 5-day-old Trichoderma culture plate and put in the center of PDA plate.The pathogen PDA plate was inoculated 48h before proceeding to the next step.The pathogen Petri dish led, and the antagonistic isolate was removed, and the two bottom plates were then joined by Parafilm.In the control, only the PDA medium was placed in the bottom plate (without the antagonistic isolate).Three replicates of the experiment were run.The tested plates were incubated for 5 days at 25 ± 2 °C.Plant pathogen mycelial diameters were measured in the control and the inverted plate and converted to an inhibitory percentage % 65 .
The inhibition percentage of the pathogen was calculated for all the tested methods using the formula: Inhibition (%) = [(rc − rt)/rc] × 100 , where rc is the radial growth of the plant pathogen on the control plate, while rt is the radial growth of the plant pathogen on the treated plate.

Gas chromatography-mass spectroscopy fractionation (GC-MS) analysis
The bioactive constituents within the cell-free supernatant of Trichoderma culture filtrate were elucidated and characterized through gas chromatography mass spectroscopy (GC-MS) analysis.For this purpose, the ethyl acetate extract was obtained as previously stated in "Effect of Trichoderma isolates against the radial growth of Alternaria pathogen in vitro" section.The resultant residues were subjected to GC-MS analysis using an Agilent 7000D instrument (Santa Clara, CA, USA) and the program conditions were programed according to Khamis et al. 66 .
Furthermore, the volatile organic compounds (VOCs) present in the Trichoderma sample were extracted using solid phase microextraction (SPME) at 40 °C for 20 min.The extracted VOCs were then injected into a GC-MS system (Agilent Technologies) equipped with a gas chromatograph model 7890B and a mass spectrometer detector model 5977A (Agilent technologies, Santa Clara, CA, USA) 67 .

Effect of Trichoderma against Alternaria leaf spot under greenhouse conditions
Based on a pot experiment conducted in a controlled greenhouse environment (temperature: 28 °C; relative humidity: 75%; photoperiod: 14 light/10 dark h), the impact of the best antagonistic isolate on the activity of the Alternaria pathogen and plant growth was assessed.Four-week-old tomato seedlings were transplanted into plastic pots (20 cm in diameter) with 1 kg of sterile soil that was evenly divided between clay, sand, and peat moss.10 mL of bioagent inoculum at a concentration of 1 × 10 7 spores/mL was sprayed into each pot after the seedlings had been transplanted for 5 days 68 .To avoid the drop of Trichoderma droplets onto the surface of the pot soil, the treated pot soil was covered when the plants were sprayed.The treatments were carried out in the greenhouse with 3 replicates and a 10 mL inoculum of the pathogen (1 × 10 5 spores/mL), as shown in Table 1.The fungicide used in this study was Ridomil Gold MZ 68%WG (4% Mefanoxam + 64% Mancozeb as active substance) at a recommended concentration of 2 g/L water.
The plants are screened for disease severity (DS) 1 month after transplantation using a 0-5 scale based on the percentage of leaf area covered by necrotic lesions 69 .After observing tomato plants, the disease incidence (DI) was determined using the formula below: The plants were also employed to evaluate the impact of the bioagent on various growth parameters, including shoot height, root length, shoot and root fresh weight (g), shoot and root dry weight (g), and total chlorophyll content, which was quantified using SPAD-502 Plus (Tokyo, Japan) 27 .

Determination of enzymes activity in tomato leaves
To prepare the tomato plant extract for enzyme assays, frozen leaves (1 g fresh mass) were ground using liquid nitrogen.Subsequently, a chilled extraction buffer consisting of 3 mL of 50 mM potassium phosphate (prepared by combining monopotassium phosphate and dipotassium phosphate, pH 7.5) was employed for material extraction.The crude extract was derived from the supernatants obtained after centrifugation of the extract at 12,000 rpm and 4 °C for 30 min.The supernatant was preserved at − 80 °C in a tube for subsequent measurement of various antioxidant enzyme activities 70 .All absorbance values were measured using a UV-1900 BMS (Waltham, MA, USA) spectrophotometer.Activity assays for peroxidase 71,72 , superoxide dismutase 73 , catalase 74 , polyphenol oxidase 75 , lipid peroxidation 76 , hydrogen peroxide 77 , total protein 70 ,and total phenolic content 78 in tomato plant leaves were conducted as per the methods detailed in the Supplementary Material.

HPLC analysis of tomato collected leaves extract
HPLC inspection was performed using an Agilent 1260 series.A column called Eclipse C18 (4.6 mm × 250 mm i.d., 5 µm) was used for the separation.The mobile phase comprises water (A) and 0.05% trifluoroacetic acid (CF 3 COOH) in acetonitrile (B), was delivered at a constant flow rate of 0.9 mL/min.Standard compounds were used for HPLC system calibration, followed by the injection of samples (5 μL) into the system at a column temperature of 40 °C.To achieve effective separation, a gradient elution program was implemented, involving distinct phases over 20 min.The separation conditions can be found in previous work 66 .The separated compounds were detected by a UV-Visible detector at 280 nm, generating a signal proportional to their concentrations.The resulting chromatogram displayed the retention time and peak area for identification and quantification.HPLC analysis confirmed the presence of target compounds in the samples at expected concentrations, with validated precision and accuracy, underscoring the method's suitability for characterizing complex mixtures and determining compound purity.

Statistical analysis
All experiments were statistically analyzed by one-way ANOVA except the data on the effect of ethyl acetate extract of different Trichoderma isolates (Tri 1-Tri 6) against pathogen growth, which was done by two-way DI% = of observed numerical rating Max.disease rating × Total number of observed plants × 100.

Ethics approval and consent to participate
This study is complied with relevant institutional, national, and international guidelines and legislation.This study does not contain any studies with human participants or animals performed by any of the authors.

Isolation and identification of the leaf spot pathogen
The fungal strain that caused tomato leaf spot symptom was found to be the same in plants that had been infected in the field.The fungal isolate was subjected to morphological and molecular analyses to determine its identity.
In order to ascertain the identity of the pathogen at the morphological level, the fungus was cultivated on a plate containing PDA medium.Subsequently, all observable characteristics were meticulously documented, including the smooth texture and the coloration, which was described as either deep or olivaceous.The conidia seen in this study were found to be solitary, exhibiting a color range from brown to olivaceous brown.These conidia were characterized by their straight or ellipsoidal tapering shape.Additionally, both transverse and longitudinal septation were observed in the conidia, as depicted in Fig. 1, panels A and B. The phenotypic traits exhibited by the pathogen provide confirmation of its classification at the genus level as Alternaria sp.
In order to validate the morphological identification, the molecular technique known as the inter-transcript spacer region (ITS) was employed.The ITS sequence obtained was subjected to a molecular identification process by blasting it through the GenBank portal.This analysis led to the identification of the isolated pathogen as Alternaria alternata Alt3, which was assigned the accession number OQ888806.
The ITS phylogenetic tree analysis revealed a complete nucleotide sequence similarity of 100% between our isolate Alt3 and A. alternata isolates with accession numbers OQ555164 and OQ560480, originating from China.The minimal level of nucleotide sequence similarity observed between A. solani fungus from China (OQ555439) and Alternaria sp.isolate from China (OQ555440) is depicted in Fig. 2.

Isolation and identification of Trichoderma isolate
Based on established taxonomic phenotypic criteria, the examination of the isolated Trichoderma isolates obtained from the roots of tomato plants exhibited morphological characteristics consistent with the attributes associated with the Trichoderma genus.The organism generated unicellular, spherical conidia that exhibited either a smooth or rough surface texture and possessed a green coloration illustrated in the Supplementary Results (Fig. S1).Solitary phialides or aggregated phialides emerged from small terminal clusters at a perpendicular angle of 90 degrees from the conidiophore on elongated, branching, and incomplete conidiophores.By amplifying PCR amplicons of the ITS primer of about 600 bp, the tef1 gene of about 400-600 bp, and the rpb2 gene amplified as a band of about 1200 bp, the Trichoderma isolate's morphological identity was verified using PCR techniques.The three amplified marker partial sequences ITS, tef1, and rpb2 were acquired and uploaded to NCBI GenBank and assigned to Trichoderma afroharzianum isolate TRI07 with accession numbers OQ820171, OR125580, and OR125581, respectively.In a comparison of the generated ITS nucleotide sequences of the TRI07 isolate using isolates from GenBank, it was shown that there was the greatest genetic homogeneity with 100% of the ITS sequence with T. afroharzianum with accession numbers (KY495202 and KY419889) from Nigeria and India, respectively (Fig. 3).Comparing the tef1 gene of the T. afroharzianum TRI07 isolate with the GenBank-identified isolates demonstrated that the highest genetic homogeneity was 100% of the tef1 sequence with T. afroharzianum with accession numbers FJ463301 and FJ463302 from Peru.Also, comparing the rpb2 gene of TRI07 isolate using isolates from GenBank-identified databases, it was shown that the highest genetic homogeneity was 100% of the rpb2 sequence with T. afroharzianum with accession numbers FJ442691 from Peru (Fig. 3).

Ethyl acetate extract of Trichoderma against pathogen mycelial growth
The data in Table 3 showed that the ethyl acetate extract of Trichoderma isolates reduced the growth of the A. alternata mycelium at concentration levels of 0, 250, 500, 1000, and 2000 µg/mL (Table 3, Fig. 5).The most suppressive bioagents against A. alternata isolate, with the highest significant inhibition growth were TRI07 (Tri 1), Tri 6, Tri 5, and Tri 2 with values of 38%, 34.96%, 33.77%, and 33.33%, respectively, while the least significant effect of inhibition percentage was 32.37% from Tri 3 and 30.88% from Tri 4.

Trichoderma isolates volatile compounds against A. alternata
In this study, the effect of volatile organic compounds (VOCs) generated from six Trichoderma isolates (Tri 1-Tri 6) against A. alternata isolate Alt3 growth was tested using the sealed plate method.The findings demonstrated that all Trichoderma isolates generated VOCs capable of inhibiting A. alternata growth (Table 4, Fig. 6).According to Table 4, T. afroharzianum isolate TRI07 (Tri 1) recorded the biggest proportion of VOCs that inhibited A. alternata growth (66.29%),followed by Tri 3 and Tri 5 with values of 65.55% and 64.07%, respectively.While  Tri 4 and Tri 6 showed moderately significant effects on VOC emissions with values of 62.96% and 60.37%, respectively.On the other side, Tri 2 had the lowest significant inhibition percentage (60.00%).
After conducting laboratory tests using various techniques, the results revealed that T. afroharzianum isolate TRI07 (Tri 1) is the most effective isolate for inhibiting A. alternata mycelium growth.Consequently, it was selected for further investigation under greenhouse conditions to assess its effects against the Alt3 isolate.

GC-MS analysis
Trichoderma afroharazianum-ethyl acetate culture filtrate Using GC-MS analysis, the bioactive components of the TRI07 isolate extract were identified.The identified components with their retention time (RT), compound names, peak areas (%), and match factor (MF) are presented in Table 5.The GC-MS analysis of the TRI07 extract's ethyl acetate filtrate revealed the presence of 13 chemical constituents (Table 5, Fig. S2), and the most abundant of them were spathulenol (28.90%), triacetin (14.03%),

Disease severity and incidence of A. alternata leaf spot on tomato plants
The disease severity (DS) and disease incidence (DI) of Alternaria alternata isolate Alt3 were recorded 30 days after transplanting seedlings.The obtained results (Table 8) showed that application of T. afroharzinum TRI07 reduced disease severity significantly compared with other treatments.Plants treated with TRI07 48 h before inoculation with Alt3 (A3) and plants treated with fungicide 48 h before inoculation with Alt3 (A5) showed the lowest level of disease severity (16.66%) with tomato leaf spot, followed by tomato plants treated with TRI07 after inoculation with Alt3 and tomato plants treated with fungicide 48 h after inoculation with Alt3 (A4, 38.88%, and A6, 33.33%, respectively).While the highest DS% was recorded on tomato plants inoculated by Alt3 only (A2, 80.55%) compared to the control plants (A1).On the other hand, A3 and A5 treatments showed the lowest value of disease incidence (44.44%).The application of A4 and A6 treatments showed the highest value of disease incidence (100%) compared to A2 treatment.

Effect of Trichoderma afroharzianum on tomato growth parameters
According to the results of the experiment conducted in a greenhouse, tomato plants in A3 treatment showed a substantial improvement (p-value ≤ 0.05) in their growth indices.The A3 treatment also significantly affected the lengths of the shoot and roots, which were measured at 71.55 cm and 32.22 cm, respectively.Tomato plants in A1 and A5 treatments had shoot and root lengths of 70.88, 30.77, 69.55, and 26.44 cm, respectively.
On the other hand, A4 and A6 treatments presented shoot and root lengths with values of (57.77 and 27.22 cm) and (63.00 and 24.66 cm), respectively.Meanwhile A3 increased the fresh and dry weight of the shoot system (13.37 and 4.16 g, respectively) compared with the control (A1) (13.51 and 4.01 g, respectively).Tomato fresh and dry shoot weight showed a high response to A5 and A6 treatments (12.45-4.03g and 12.20-3.55g, respectively).While A2 treatment showed the lowest fresh and dry weights of the tomato shoot system (11.27 and 3.25g, respectively).
For fresh and dry weight of tomato root (Table 9), plants in A3 treatment showed a highly significant response (11.71 and 1.98 g, respectively), and A5 treatment (11.64 and 1.84g, respectively) compared to control (A1).Furthermore, the lowest values were reported with A2 treatment (8.73 and 1.17 g, respectively).As compared to control (A1), the A3 treatment significantly improved the chlorophyll content (25.46 SPAD units), followed by the A5 and A6 treatments, with SPAD units of 22.12 and 20.55, respectively (Table 9).A4 treatment, with a 19.9 SPAD value, demonstrated moderate significance, while A2 treatment demonstrated the lowest significance (10.5 SPAD value).

Antioxidant enzymes activities
Four antioxidant enzymes, namely catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and polyphenol oxidase (PPO), were distinguished upon A. alternata infection, Trichoderma, and fungicide treatments (Table 10).When compared to the control plant (A1), the A. alternata treatment (A2) had the greatest CAT activity value (0.15 µM/g FW), followed by the A3 treatment (0.089 µM/g FW).Contrarily, A4 treatment reduced CAT activity (0.074 µM/g FW) compared to control, which was then followed by A5 and A6 treatments with values of 0.034 and 0.05 µM/g FW, respectively.In terms of POD activity, the A3 treatment value was 1.24 µM/g FW, followed by the A2 treatment value of 1.04 µM/g FW, as opposed to the control (A1) with a value of 0.56 µM/g FW.The lowest value was shown with A5, 0.28, and A6, 0.31 µM/g FW, while the moderate value was obtained from the A4 treatment value of 0.51 µM/g FW.The tomato plants in A3 treatment exhibited the highest value of SOD activity (0.51 µM/g FW), followed by A4 treatment, 0.44 µM/g FW.Additionally, tomato plants in A6 showed a small increase in SOD activity (0.38 µM/g FW) greater than tomato plants in A4 or the healthy control plant (A1), which was not treated with a fungicide.The best value of induced PPO activity was 0.75 µM/g FW in A6 compared to the A. alternata treatment (A2).Additionally, compared to the control (A1), the PPO activity in the A3, A5, and A4 treatments was somewhat higher at 0.46, 0.45, and 0.43 µM/g FW, respectively (Table 10).10).

Total protein assay
Tomato plants in A3 treatment showed the highest protein content, reaching 333.56 µg/mL, followed by A1 and A5 treatments (332.68 and 329.52 µg/mL, respectively).Furthermore, there was no significant difference between tomato plants in the A4 treatment (326.71 µg/mL) and the A2 treatment (326.71 µg/mL).On the other hand, A6 plants showed a slight increase in the total protein content of leaves (316.36 µg/mL) as shown in Table 10.

Phytochemicals in tomato leaf extract
Table 11 and Fig. S3 show the ethanolic extract HPLC chromatograms for the six different tomato plant treatments.Based on the HPLC results, the different treatments A1, A2, A3, A4, A5, and A6 had different amounts of polyphenolic compounds: 2770.Other compounds like naringenin and quercetin as flavonoid compounds (µg/g) were overexpressed in A2 plants with an accumulation value of 95.39 and 21.18, respectively, compared to A1 plants (26.39 and 13.24).In addition to catechin, rutin and vanillin compounds were detected in the A3 treatment at high concentrations (50.59, 6423.57, and 65.77, respectively).

Discussion
Alternaria leaf spot typically affects many vegetables, including tomato plants.One fungal isolate of the tomato leaf spot pathogen, identified as Alternaria alternata Alt3, was investigated.Using Simmons' morphological characteristics, including colony morphology, size, conidial form, and septation pattern of conidia, the fungus strain was identified morphologically 79 .Based on ribosomal internal transcribed spacer (ITS) DNA sequence analysis, PCR was recently utilized to detect Alternaria spp. in tomato samples 43 .Therefore, depending on morphological characters and amplification and sequencing of ITS region, the pathogen was identified as A. alternata 80 .
According to the morphological characteristics of the Trichoderma isolates and genetical characterization, the most effective isolate was identified using the ITS, tef1, and rpb2 genetic markers as Trichoderma afroharzianum isolate TRI07.Within Trichoderma species complexes, it has been demonstrated that ITS sequence analysis is poor at separating closely related species 81 .Tef1 and rpb2 gene sequences are particularly instructive and have been demonstrated to be helpful in examining closely related strains at the species level 82,83 and popular markers for classifying Trichoderma strains 84 .Chemical fungicides are an efficient way to control the pathogen A. alternata.However, it might not always be appropriate to use fungicides to manage fungal infections in plants.Due to the hazards involved, chemical control of plant diseases is waning in popularity 85 .However, biocontrol agents are an environmentally friendly and sustainable replacement for dangerous fungicides 86 .
Trichoderma species are considered one of the most significant biological pest controllers because they reduce plant diseases through a variety of methods, including competition, mycoparasitism, antibiosis, and the induction of systemic resistance 87 .One of the most effective Trichoderma fungi for use as plant disease-suppressing inoculants is T. afroharzianum 88 .
In our in vitro studies we used three techniques to test the inhibition for mycelial growth of A. alternata like dual culture technique, food poison technique and volatile organic compounds (VOCs) which showed that T. afroharzianum was the most effective isolate to inhibit A. alternata fungus.In the initial dual culture assessments conducted in this investigation, T. afroharzianum TRI07 exhibited a growth rate three times higher than that of the pathogen.This superior growth rate provides TRI07 isolate with a significant competitive advantage for space and nutrients compared to A. alternata.During dual in vitro confrontations, TRI07 isolate not only suppressed the growth of the pathogens but also engaged in mycoparasitism against the pathogen.This mycoparasitic mechanism was corroborated through microscopic images of the confrontation assays.Microscopic examinations indicated that T. afroharzianum deployed a multifaceted mechanism against the tested A. alternata pathogen.This strategy involved hyper-sporulation, conidial binding to various structures of pathogenic fungi, and swift dissemination within the microenvironment of pathogens.Trichoderma surrounded the pathogen's hyphae, depriving them of nutrients.The engagement of lytic enzymes played a crucial role in this mechanism, inducing morphological alterations and lysis of the pathogen's conidia, thereby promoting mycoparasitism.The intricate process of direct fungal attack (mycoparasitism) involves sequential events of recognition, attack, and subsequent penetration and killing of the host 89 .Similar outcomes were observed in studies involving Trichoderma strains, where their conidia enveloped those of Botrytis cinerea, inhibiting germination and subsequent pathogen development, and morphological changes were also documented in Fusarium sp. 90,91.The attack on pathogenic fungi involving conidia and coiling hyphae could be prompted by organic compounds and/or nutrient gradients emanating from the pathogens and sensed by Trichoderma 92 .Meanwhile, Trichoderma harzianum and T. viride strains performed best in a dual culture experiment against A. alternata with inhibition percentages of 75.04 and 67.83%, respectively 93 .Additionally, the hyphal penetration of T. harzianum into the hyphae of A. alternata, F. proliferatum and B. sorokiniana was demonstrated to be another mycoparasitic mechanism of action 94,95 .
The current study revealed that the ethyl acetate extract of all Trichoderma species efficiently suppressed the growth of the pathogen.The Trichoderma species are recognized for their ability to generate several secondary The identification of bioactive constituents in the ethyl acetate extract of T. afroharazianum was conducted through the application of gas chromatography-mass spectrometry (GC-MS) analysis.This analytical technique was employed to detect volatile organic compounds (VOCs) and non-volatile organic compounds (non-VOCs) present in the culture filtrates of T. atroviride and T. asperellum, respectively 97 .The ethyl acetate analysis revealed several compounds present in the T. afroharzianum, with significant proportions represented by specific constituents.Among these, spathulenol was the predominant compound, constituting 28.90% of the total area.Triacetin and aspartame followed, contributing 14.03% and 12.97%, respectively.Notably, oleic acid, β-lonone, and 13-methylpentadecanoic acid methyl ester were also identified as substantial components, each accounting for 6.39%, 5.82%, and 5.63% of the area, respectively.Moreover, ethylene brassylate, strychane, 1-acetyl-20à-hydroxy-16-methylene, 2,3,4,5-tetrahydroxypentanal, o-desmethylvenlafaxine, cyclobarbital, and 1-tetradecanol were detected in varying proportions, each contributing to the overall chemical composition of T. afroharzianum extract.The high percentage of spathulenol is noteworthy due to its reported biological activities, including antimicrobial properties.In other study, the primary compound identified in the essential oil of Baccharis dracunculifolia was spathulenol, an oxygenated sesquiterpene characterized by high hydrophobicity.The antimicrobial activity of sesquiterpenes, including spathulenol, is often attributed to a cell membrane-disrupting mechanism, leading to the release of K + ions from pathogen cells, which increase the permeability across the plasma membrane, facilitating interaction with intracellular proteins and/or intra-organelle sites 98,99 .With a surface polarity of 20.2 Å 2 , spathulenol possesses one hydrogen bond donor and one acceptor 100 .According to Pajouhesh and Lenz 101 , compounds with a polar surface area (PSA) of 60 Å 2 or less are fully absorbed by the cell, indicating the ability of spathulenol to traverse cellular membranes.The observed discrepancies in efficacy could perhaps be attributed to the presence of spathulenol, β-phellandrene, germacrene D, bicyclogermacrene, and 1,8-cineole in the specimens from Mpumalanga.These constituents have previously been identified as the primary chemicals in oils that exhibit antifungal properties 102,103 .Triacetin, one of the minor compounds, also has antifungal activity 104 .A study conducted to explore alternative strategies for addressing the presence of major plant disease pathogens, including Pythium ultimum, Rhizoctonia solani, Crinipellis perniciosa, and Pyrenophora avenae and their findings, as reported by Walters et al. 105 , suggest that linolenic, linoleic, and oleic acids could potentially offer beneficial effects in this regard.Tychonema bourrellyi culture filtrates contained a significant amount of β-ionone, which may be the source of the organism's allelopathic biocidal activity 106 .
The results of our investigation indicate that T. afroharazianum emitted a total of 34 VOCs in response to A. alternata in liquid culture.Notably, the VOCs identified were 1-octen-3-one, 2H-pyran-2-one, 6-pentyl, n-hexadecanoic acid, and hexanedioic acid, bis (2-ethylhexyl) ester.These compounds exhibited significant antifungal properties when tested in vitro.The chemical known as acetophenone was shown to possess antifungal properties in laboratory conditions when tested against Penicillium italicum.This discovery was made among a group of VOCs produced by T. afroharzianum T22, as reported by Li et al. 107 .The VOCs produced by T. harzianum exhibited significant (p < 0.05) antifungal activity against Botrytis cinerea, A. panax, Cylindrocarpon destructans, and Sclerotinia nivalis.Joo et al. 108 also conducted experiments to evaluate the effects of the treatment on plant growth promotion, repression, and enhancement.
A recent study conducted by Phoka et al. 109 revealed that the endophytic fungus T. asperelloides PSU-P1 produces notable VOCs that contribute to its antifungal activities.Specifically, the identified VOCs were 2-methyl-1-butanol and 6-pentyl-2H-pyran-2-one.The development of Colletotrichum gloeosporioides and A. alternata is inhibited by the T. koningiopsis T2 strain through the production of metabolites derived from VOCs and non-VOCs 110 .The presence of bioactive chemicals in the extract suggests that the VOCs emitted by T. harzianum have a significant role as both an antioxidant and an antifungal agent, as indicated by Lakhdari et al. 111 .
In the greenhouse study, the highest DS% and DI% values were found in the A3 treatment in vivo tests with tomato plants treated with T. afroharzianum 48 h before inoculation with A. alternata.These findings are consistent with Chowdappa et al. 112 who reported that B. subtilis and T. harzianum inhibited the growth of P. infestans and A. solani in vitro.As reported by Elsherbiny et al. 113 T. afroharzianum significantly enhanced the growth of tomato plants, including plant height, number of leaves/plant, dry weight, and root activity, through colonization in the rhizosphere and root system.T. afroharzianum also increased tomato growth parameters.For instance, T. afroharzianum strain T22, the active ingredient of a commercial biofungicide product 114 was found to enhance plant growth of significant horticultural crops, such as tomatoes, peppers, lettuce, ornamentals, and woody crops, and to control diseases under field and greenhouse conditions [115][116][117] .Through effective colonization in the plants' rhizosphere and root system, T. afroharzianum TM2-4 considerably accelerated the growth of tomato plants in terms of dry weight, number of leaves per plant, plant height, and root activity 118 .
According to our study, T. afroharizum enhanced defense and detoxifying mechanisms, resulting in quick and effective responses to pathogen inoculation.Additionally, the activities of phenolics, flavonoids, total protein, CAT, SOD, and PPO may be crucial for tomato survival when under stress from fungi.Reactive oxygen species (ROS) are produced when a plant experiences stress and can affect plant growth by damaging DNA, proteins, and membrane systems 119 .The ability of enzymes like catalase and superoxide dismutase (SOD) to "clear out" ROS is reflected in their enzyme activity, which indirectly indicates the plant's capacity to combat and reduce ROS 120 .High activities of POD and SOD were seen in a mutant soybean (Glycine max) that induces expression of the Tachi gene from T. asperellum.This mutant exhibited higher resistance to S. sclerotiorum 121 .Furthermore, T. harzianum inoculation markedly boosted the activity of POD, CAT, and PPO in squash 122 .Additionally, T.  123 .Secondary metabolites, such as vanillic, chlorogenic, and caffeine acids, were present in tomato leaves than in red fruits.The findings imply that inedible tomato plant components can be exploited as a source of raw materials for antioxidants, anti-inflammatory drugs, fungistats, and insecticides 124 .Trihydroxybenzoic acid, or gallic acid, is a naturally occurring polyphenol chemical that is present in a variety of plant species and has been proven to have antifungal and antibacterial activities 125 .
In our recent work, we found that the utilization of T. pubescens either independently or in combination with other interventions for the management of plant pathogen infections led to elevated concentrations of phenolic acids, including chlorogenic and coumaric acids 57 .In summary, treating plants with T. afroharzianum yielded positive outcomes in terms of antioxidant response and plant health.Enhanced production of antioxidant enzymes (CAT, POD, SOD, PPO) demonstrated improved defense mechanisms.However, infected plants showed increased MDA and H 2 O 2 levels, indicating diseaserelated cellular damage.T. afroharzianum A3 treatment also led to higher levels of phenolics, known for their antioxidant properties.These findings suggest that T. afroharzianum has the potential to serve as an effective biocontrol agent against leaf spot disease, ultimately leading to enhanced post-crop productivity and improved plant health.
In light of recent reports indicating that T. afroharzianum has been associated with causing diseases in plants such as maize 126 , and there have concerns about its potential impact on other plant cultivars, it is crucial to address the potential limitations and risks associated with the use of this biocontrol agent.While our study focuses on the positive aspects of T. afroharzianum in controlling Alternaria leaf spot in tomato, it is essential to recognize the need for further investigation into its potential effects.The comment raises valid concerns regarding the genetic diversity within the T. afroharzianum cluster, suggesting that variations in behavior among different strains may be attributed to genetic differences.This observation underscores existing knowledge gaps in understanding the genetic intricacies of T. afroharzianum.Consequently, it becomes imperative to conduct comprehensive genetic studies to unravel strain-specific characteristics and shed light on their varied pathogenic behaviors.Another critical aspect to consider is the influence of environmental factors and host-specific interactions.Regional climate variations and diverse plant cultivars may contribute to varying responses in Trichoderma behavior.Recognizing the multifaceted nature of these interactions is essential for interpreting contradictory findings in studies conducted in different regions and under different environmental conditions.The complexity of studying Trichoderma behavior necessitates a thorough consideration of methodological variations.Differences in experimental protocols between studies may contribute to conflicting outcomes.To address this, it is crucial to acknowledge these methodological nuances and advocate for standardized approaches.Comparative studies that align methodologies will be instrumental in validating and reconciling diverse research findings.The observed pathogenicity of certain Trichoderma strains raises questions about their application as biocontrol agents.Conducting a comprehensive risk-benefit analysis becomes paramount.While recognizing the potential benefits of Trichoderma in biocontrol, it is equally important to assess and communicate the associated risks.This cautious and well-informed approach is critical for determining the suitability of Trichoderma strains in agricultural practices.
Finally, future research and field trials should delve into the comprehensive assessment of T. afroharzianum safety and potential limitations in practical agricultural applications.

Conclusions
Alternative environmentally acceptable bioagents are required to control plant diseases in the agriculture sector.Our research demonstrated the impact of T. afroharzianum on the in vitro and greenhouse-induced leaf spot disease caused by A. alternata.In the experimental greenhouse, T. afroharzianum displayed a highly significant increase in shoot and root length, shoot and root fresh and dry weight, and chlorophyll contents.The growth of A. alternata in vitro was greatly slowed down using dual culture techniques, volatile organic compounds, and Trichoderma extract.The GC-MS analysis for VOCs of T. afroharzianum with A. alternata showed increasing compounds like 1-octen-3-one, 2H-pyran-2-one, 6-pentyl, n-hexadecanoic acid and hexanedioic acid, bis (2-ethylhexyl) ester.T. afroharzianum treatment exhibited an increase in the synthesis of antioxidant enzymes (CAT, POD, SOD, and PPO) after 21 days of inoculation in vivo, and high MDA and H 2 O 2 levels were found in the infected plants.The HPLC results of T. afroharzianum A3 treatment showed increasing in gallic, chlorogenic, caffeic and coumaric acids.To prevent the leaf spot disease and post crop productivity, T. afroharzianum causes systemic resistance, inhibits the growth of A. alternata, and fosters the development of tomato plants.
Trichoderma afroharzianum TRI07 stands out due to its potent antagonistic effects against various plant pathogens, showcasing a higher efficacy compared to other strains within the Trichoderma genus.Its distinct ability to enhance plant growth and induce systemic resistance makes it a promising candidate for biocontrol.Existing control methods often face limitations such as environmental concerns, resistance development, and inconsistent efficacy.TRI07's unique features, including enhanced tolerance to environmental stressors, contribute to its potential as a sustainable alternative to conventional chemical approaches.This study innovatively explores TRI07's molecular mechanisms, emphasizing its genetic attributes that contribute to heightened biocontrol capabilities.By dissecting these mechanisms, the research aims to develop more targeted and effective biocontrol strategies, overcoming the shortcomings of current methods.This study offers a sustainable strategy for suppressing Alternaria leaf spots, a significant pathogen affecting tomato crops.The approach relies on biological control and has the potential to be integrated with other environmentally friendly practices, serving as an alternative to chemical fungicides in various processes.

Figure 2 . 2 Figure 3 .
Figure 2. A phylogenetic tree was generated using the eight most parsimonious ITS sequences in comparison to the isolated Alternaria alternata Alt3 (OQ888806) sequence.

Figure 6 .
Figure 6.Effect of volatile organic compounds (VOCs) emitted from Trichoderma isolates (Tri 1-Tri 6) against A. alternata in vitro using the inverted sealed plate method.

Figure 8 .
Figure 8. Volatile organic compounds (VOCs) emitted from the interaction between T. afroharazianum TRI07 and A. alternata Alt3 conducted by solid phase microextraction method.

Table 1 .
The treatments used in the greenhouse experiment.

Table 2 .
The effectiveness of Trichoderma isolates on A. alternata growth using dual culture plates.*Values are means ± standard deviation.If there are distinct letters next to the data values for inhibition%, it means that the values were significantly different at p-value ≤ 0.05.

Table 5 .
Identified compounds in ethyl acetate extract of Trichoderma afroharzianum TRI07 defined by GC-MS.RT retention time, MF molecular formula.

Table 7 .
Volatile organic compounds (VOCs) produced from interaction between T. afroharazianum TRI07 and A. alternata Alt3 by solid phase microextraction method.RT retention time.

Table 8 .
Effect of Trichoderma isolates on A. alternata disease severity (DS%) and disease incidence (DI%).A1 tomato plant control, A2 plants inoculated by A. alternata (Alt3) only, A3 plants inoculated with T. afroharzinum TRI07 48 h before inoculation with Alt3, A4 plants inoculated with TRI07 after 48 h inoculation with Alt3, A5 plants treated with fungicide 48 h before inoculation with Alt3, A6 plants treated with fungicide after 48 h of inoculation with Alt3.*Values are means ± standard deviation.The presence of different letters next to the data values in each column indicates that the differences were significant at p-value ≤ 0.05.

Table 9 .
Vegetative parameters and chlorophyll content of tomato plants in vivo.A1 tomato plant control, A2 plants inoculated by A. alternata (Alt3) only, A3 plants inoculated with T. afroharzinum TRI07 48 h before inoculation with Alt3, A4 plants inoculated with TRI07 after 48 h inoculation with Alt3, A5 plants treated with fungicide 48 h before inoculation with Alt3, and A6 plants treated with fungicide after 48 h of inoculation with Alt3.The presence of different letters next to the data values in each column indicates that the differences were significant at p-value ≤ 0.05.*Values are means ± standard deviation.Oxidative stress markers assayThe current findings on tomato plants demonstrate the importance of H 2 O 2 and MDA as indicators of oxidative stress.In the MDA results, the treatments (A5) and (A6) were 0.66 and 0.61 µM/g FW, compared to the healthy tomato plants (A1, 0.48 µM/g FW).While A4 displayed a low value (0.46 µM/g FW) compared to the control, A3 displayed a slightly higher value (0.53 µM/g FW) in MDA content compared to the A2 treatment.At the same time, the H 2 O 2 assay revealed that A2 treatment had the maximum amount (46.63 µM/g FW) in plants, followed by A3 treatment (37.70 µM/g FW).Also, A4, A5, and A6 treatments encouraged a drop in H 2 O 2 concentration with values of 4.66, 6.63, and 4.95 µM/g FW, respectively (Table Vol:.(1234567890) Scientific Reports | (2024) 14:1874 | https://doi.org/10.1038/s41598-024-52301-2www.nature.com/scientificreports/

Table 10 .
The activities of CAT, POD, SOD, PPO, MDA and H 2 O 2 enzymes, total protein and total phenolic compounds (TPC) in tomato plant leaves under greenhouse study.A1 tomato plant control, A2 plants inoculated by A. alternata (Alt3) only, A3 plants inoculated with T. afroharzinum TRI07 48 h before inoculation with Alt3, A4 plants inoculated with TRI07 after 48 h inoculation with Alt3, A5 plants treated with fungicide 48 h before inoculation with Alt3, and A6 plants treated with fungicide after 48 h of inoculation with Alt3.The presence of different letters next to the data values in each column indicates that the differences were significant at p-value ≤ 0.05.*Values are means ± standard deviation.

Table 11 .
96tected polyphenolic compounds using HPLC analysis in tomato leaf extracts.A1 tomato plant control, A2 plants inoculated by A. alternata (Alt3) only, A3 plants inoculated with T. afroharzinum TRI07 48 h before inoculation with Alt3, A4 plants inoculated with TRI07 after 48 h inoculation with Alt3, A5 plants treated with fungicide 48 h before inoculation with Alt3, and A6 plants treated with fungicide after 48 h of inoculation with Alt3, ND not detected..The literature supports similar conclusions, Murtaza et al.96examined the antifungal properties of five Trichoderma species, specifically T. viride, aureoviride, reesei, koningii, and harzianum, against Alternaria citri.The culture filtrate of T. harzianum exhibited remarkable efficiency in suppressing the growth of the investigated fungal species, achieving a suppression rate of up to 93%.The fractionation-guided bioassay of T. harzianum metabolites revealed a significant 68% reduction in the growth of A. citri when exposed to a 1% concentration of ethyl acetate fraction. metabolites afroharzianum elevated CAT activity and decreased H 2 O 2 content Vol.:(0123456789) Scientific Reports | (2024) 14:1874 | https://doi.org/10.1038/s41598-024-52301-2www.nature.com/scientificreports/